Vascular endothelium constitutes the innermost lining of blood vessels. In this key anatomic position it may play an essential role in a variety of pathophysiological processes, including thrombosis, inflammation, immune responses, and tumor cell metastasis. In particular, vascular endothelium has been shown to play an active role in the adhesion of blood leukocytes (neutrophils, monocytes, and lymphocytes), presentation of antigen to lymphocytes, and contribution to the binding of nonlymphoid tumor cells. Recent evidence suggests that specific endothelial cell surface molecules are involved in these processes. For example, endothelial-leukocyte adhesion molecule-1 (ELAM-1) is an inducible M.sub.r 115,000 glycoprotein found on the surface of activated endothelial cells, that mediates the adhesion of blood neutrophils. Through this function, ELAM-1 appears to play a central role in mediating neutrophil influx into sites of acute inflammatory processes. The invention described herein (monoclonal antibody E1/6 and the glycoprotein recognized by the antibody) relates functionally to the attachment of both normal and neoplastic lymphocytes, and certain nonlymphoid tumor cells (e.g. melanoma) to endothelium.
In the development of an immune response, a foreign antigen must be processed and "presented" to lymphocytes in combination with MHC molecules by a second cell designated as "antigen presenting". A number of cell types have been implicated in the process of antigen presentation. Experimental evidence suggests that certain dendritic cell populations of lymphoid tissue and elsewhere, endothelial cells, and macrophages can function as antigen presenting cells. Studies of antibody binding using human tissues and an immunoperoxidase technique reveal that E1/6 antigen is expressed on endothelium, dendritic cell populations, and in certain tissue macrophages (e.g. Kupffer cells of the liver), as well as Bowman's epithelium of the kidney. Given such a distribution, and function as a lymphoid cell adhesion molecule, E1/6 antigen may play a key role in a variety of inflammatory and immunological responses (e.g. antigen presentation and graft rejection). As further described below, the ability of antibody E1/6 to block tumor cell adhesion to endothelium also suggests a role in the process of hematogenous metastasis.
Endothelium, the cells which line blood vessels and lymphatics, constitutes the first anatomic barrier between circulating tumor cells (and tumor subpopulations) and extravascular tissue (a process called extravasation). Thus, adhesion of tumor cells to the endothelium plays a role in hematogenous metastasis, a multi-stage phenomenon which includes the distribution and interaction of blood-borne tumor cells with blood vessel walls. Although various tumor cells have been shown to adhere to endothelial monolayers in vitro, early observations indicated that the subendothelial matrix, a part of the microvasculature, is generally more adhesive for tumor cells than unstimulated endothelial cells themselves.
In vivo, localized modulation of the surface adhesive properties of vascular endothelium may influence the first step in tumor implantation, thus potentially affecting the incidence or pattern of hematogenous metastasis. Hematogenous metastasis is in itself an inefficient process, with the great majority of tumor cells failing to survive the circulation, in part due to the endothelium's ability to act as a barrier to extravasation.
However, in vivo observations indicate that certain tumor cells directly adhere to intact endothelium, and subsequently transmigrate to contact the basal lamina, often after many hours. In short, the ability of circulating tumor cells to traverse the vascular endothelial lining may be a key factor in determining the ultimate survival and metastatic capacity of the tumor cell.
Focal adhesion of blood-borne tumor cells and blood leucocytes to the vascular lining is a key step in a variety of pathophysiological processes, including hematogenous metastasis, inflammation, tissue repair, and other pathophysiological processes involving endothelial activation. Bacterial endotoxin and multifunctional (inflammatory/immune) cytokines, such as interleukin-1 and tumor necrosis factor (TNF), can act directly on cultured human endothelial cell (HEC) monolayers, in a time- and protein synthesis-dependent fashion, to increase the adhesion of certain human melanoma and carcinoma cell lines. These mediators can act directly on HEC monolayers to increase the adhesion of blood leukocytes and leukocyte cell lines. This effect is mediated, at least in part, through the synthesis and expression of specific endothelial cell surface proteins that bind leukocytes.
Vascular endothelial cells of various organs preferentially bind certain tumor cells, and display different cell surface components.
Recent studies suggest that activation of cultured human umbilical vein endothelial cells (HEC) also increase the adhesion of certain other cancer cells, such as carcinoma cell lines, suggesting that E1/6 protein or similar molecules may play an important role in the metastasis of many cancer cell types.
Since the expression of E1/6 protein can be modulated by soluble mediators, localized and transient changes in expression may occur in a variety of pathophysiological processes. In addition, the production within a vessel of cytokines by tumor cells themselves could potentially lead to upregulation of adhesion molecules on adjacent endothelium, thereby influencing extravasation, the discharge of blood cells outside the vascular sytem.
E1/6 protein is expressed by vascular endothelium in human lung, skin, brain and lymphoid tissue in vivo, and its increased expression is evident in sites of certain pathophysiological processes involving endothelial activation. E1/6 protein appears to play a major role in melanoma-endothelial cell adhesion and thus, may contribute to the development and distribution of metastatic lesions.
Endothelial cell surface properties can be modulated by a number of stimuli in vitro, including coagulation factors (e.g. thrombin), inflammatory/immune cytokines, and extracellular matrix components. Activation of endothelium by interleukin-1 (IL-1) and tumor necrosis factor (TNF) induces the biosynthesis and expression of cell surface molecules that mediate the adhesion of blood leukocytes. Endothelial-leukocyte adhesion molecule-1 (ELAM-1), a M.sub.r 115,000 glycoprotein expressed by activated endothelium, acts as a receptor for polymorphonuclear leukocytes. Intercellular adhesion molecule-1 (ICAM-1) is a M.sub.r 90,000 glycoprotein expressed by both leukocytic and non-leukocytic cell types and contributes to a variety of cell--cell adhesive interactions including lymphocyte aggregation, lymphocyte-fibroblast adhesion, and leukocyte-endothelial adhesion.
Monoclonal antibodies (Mab), that bind to ELAM-1 were generated against IL-1 or TNF-stimulated HEC and were found to react with the same inducible cell surface protein (Mr 115,000) present on cytokine-stimulated HEC, but not on unstimulated HEC. One of these Mab, H18/7, significantly inhibits the adhesion of human neutrophils and HL-60 cells to stimulated HEC, supporting the designation endothelial-leukocyte adhesion molecule-1 (ELAM-1). A full length cDNA encoding ELAM-1 has been isolated by transient expression in COS cells. Cells transfected with this cDNA express a surface glycoprotein recognized by Mab H4/18 and H18/7 and support the adhesion of neutrophils and HL-60 cells.
ELAM-1 is an inducible endothelial cell surface glycoprotein structurally related to complement regulatory protein (e.g. complement receptor 1) and to lectin-like molecules (e.g. asaialoglycoprotein receptor). The expression of ELAM-1 is essentially restricted to activated endothelium. ICAM-1, a member of the immunoglobulin supergene family, is found at low levels on unstimulated endothelium in vitro and in vivo. ICAM-1 is also present on a variety of lymphoid and nonlymphoid cell types. Assessment of potential relationships between E1/6 protein and ELAM-1 or ICAM-1 awaits determination of its primary structure. Two other endothelial adhesion molecules, the vascular addressing, are constitutively expressed on high endothelial venules of lymphoid tissues and play a role in lymphocyte homing. These addressins may be representative of organ specific structures postulated to be involved in tumor metastasis. However, to date, organ specific molecules that mediate adhesion of nonlymphoid tumor cells have not been identified.
Monoclonal antibody E1/6 was first observed to block endothelial cell-melanoma cell adhesion, whereas monoclonal antibodies to two other inducible molecules, ELAM-1 and ICAM-1, had no significant effect.
In short, functional and immunochemical characterizations show that E1/6 mAb and antigen are distinct from ELAM-1, ICAM-1, and ICAM-2.